Prostate cancer is the most common cancer in men in Europe and North America (Crawford (2003) Urology 62: 3-12; Gronberg et al. 92003) Lancet 361: 859-864; Pienta et al. (2006) Urology 48: 676-683). One of the clinical diagnosis tools for prostate cancer is the measurement of plasma protein concentration of the prostate-specific antigen (PSA or hK3), which is a member of the large kallikrein (hK) protease family (for reviews, see, e.g., Yousef and Diamandis (2001) Endocr. Rev., 22: 184-204; Denmeade and Isaacs (2002) Nat. Rev. Cancer 2: 389-396; Denmeade and Isaacs (2004) BJU Int 93 Suppl 1: 10-15) normally secreted from prostate luminal epithelial cells. Unlike other kallikrein family members, PSA is a chymotrypsin-like serine protease (Robert et al. (1997) Biochemistry 36: 3811-3819). In prostate cancer, PSA, aided by the proteolytic activity, is involved in tissue remodeling against the extracellular matrix, contributing critical control mechanisms to tumor invasion or progression. Other proteases play similar roles in cancers as well.
The introduction of plasma PSA screening since the 1980s has greatly improved the diagnosis, staging, and management of prostate cancer (Denmeade and Isaacs (2002) Nat. Rev. Cancer 2: 389-396); however, measurement of plasma PSA concentration does not differentiate the prostate cancer patients from those with benign prostatic hyperplasia, leading to a high false positive rate, requirement for more expensive biopsies, and even unnecessary surgical procedures (Denmeade and Isaacs (2004) BJU Int 93 Suppl 1: 10-15; Robert et al. (1997) Biochemistry 36: 3811-3819). Efforts to enhance the clinical value of the PSA for early detection of prostate cancer have included the characterization of various molecular isoforms of PSA (Mikolajczyk et al. (2004) Clin. Chem., 50: 1017-1025; Mikolajczyk and Rittenhouse (2003) Keio J. Med. 52: 86-91; Mikolajczyk et al. (2004) Clin. Biochem. 37: 519-528). Among those various isoforms, the proteolytically active subpopulation of PSA is accepted as a more useful tumor marker and malignancy predictor than the serum PSA concentration (Wu et al. (2004) Prostate 58: 345-353; Wu et al. (2004) Clin. Chem., 50: 125-129). Simple detection of the presence of PSA by a traditional immunostaining method can not reveal the proteolytic activity of PSA; therefore, it is of great importance to develop new methods to discriminate the proteolytically active isoform. Seminal fluid has been demonstrated to carry an abundance of proteolytically active PSA and is a biological source of PSA for protease activity assays (Brillard-Bourdet et al. (2002) Eur. J. Biochem., 269: 390-395; Rehault et al. (2002) Biochim. Biophys. Acta 1596: 55-62). The concentration of proteolytically active PSA in seminal fluid is at 10-150 μM (Rehault et al. (2002) Biochim. Biophys. Acta 1596: 55-62), while its concentration in the plasma is much lower, from less than 0.1 nM in healthy individuals to higher than 1 nM in patients with prostate disease (Rittenhouse et al. (1998) Crit. Rev. Clin. Lab. Sci., 35: 275-368). However, an assay that measures the proteolytic activity of PSA in seminal fluid or biopsy samples from fine needle aspiration is still not widely accepted, due to the quick decay of the proteolytic activity, and the limited amount of seminal fluid available from old patients or biopsy samples.
The sensitivity of current detection methods reach subnanomolar concentrations for PSA protein (Acevedo et al. (2002) Clin. Chim. Acta 317: 55-63; Charrier et al. (1999) Electrophoresis 20: 1075-1081; Bjartell et al. Prostate Cancer P D 2: 140-147) (mostly determined by the binding affinity of the antibody to PSA), and relatively large sample volume (milliliter) is required. However, the enzymatic assays have not enjoyed the same sensitivity enhancement.